Abstract
Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.
Highlights
The Pasteurellaceae family consists of several genera, of which Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium are regularly identified during routine health monitoring of laboratory mice
We assessed the suitability of the assay to distinguish between different Pasteurellaceae species and strains by testing the reference strains obtained from different culture collections or isolated from mice within our facility
We described a new approach using a real-time PCR/high-resolution melting curve analysis (HRM) system for identification and discrimination of Pasteurellaceae species, one of the most prevalent bacterial pathogens found in laboratory mice
Summary
The Pasteurellaceae family consists of several genera, of which Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium are regularly identified during routine health monitoring of laboratory mice. Pasteurellaceae constitute the most prevalent bacterial pathogens in experimental facilities worldwide [1,2]. The pathogenicity of most Pasteurellaceae species is low, P. pneumotropica is associated with variable clinical manifestations such as infections of the eye, genital tract, and respiratory system [3]. Even subclinical P. pneumotropica infections in immunocompetent mice could represent an unwanted experimental variable and may influence results [4]. The Federation of European Laboratory Animal Science Associations revised their recommendations on Pasteurellaceae reporting. While the prior version recommended monitoring all Pasteurellaceae species, specific identification of P. pneumotropica is currently recommended [5]. Depending upon the research focus, some facilities may include identification of other
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